Myotubularin-related protein (MTMR) 9 determines the
enzymatic activity, substrate specificity, and role in
autophagy of MTMR8
Jun Zoua,1, Chunfen Zhangb,1,2, Jasna Marjanovicc, Marina V. Kisselevab, Philip W. Majerusb,d,2, and Monita P. Wilsonb,2
aDepartment of Pathology and Immunology,bDivision of Hematology, Department of Internal Medicine, anddDepartment of Biochemistry and Molecular
Biophysics, Washington University School of Medicine, St. Louis, MO 63110; andcDivision of Basic and Pharmaceutical Sciences, St. Louis College of Pharmacy,
St. Louis, MO 63110
Contributed by Philip W. Majerus, May 1, 2012 (sent for review February 24, 2012)
The myotubularins are a large family of inositol polyphosphate
3-phosphatases that, despite having common substrates, subsume
unique functions in cells that are disparate. The myotubularin
family consists of 16 different proteins, 9 members of which
possess catalytic activity, dephosphorylating phosphatidylinositol
3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphos-
phate [PtdIns(3,5)P2] at the D-3 position. Seven members are in-
active because they lack the conserved cysteine residue in the
CX5R motif required for activity. We studied a subfamily of homol-
ogous myotubularins, including myotubularin-related protein 6
(MTMR6), MTMR7, and MTMR8, all of which dimerize with the
catalytically inactive MTMR9. Complex formation between the ac-
tive myotubularins and MTMR9 increases their catalytic activity
and alters their substrate specificity, wherein the MTMR6/R9 com-
plex prefers PtdIns(3,5)P2 as substrate; the MTMR8/R9 complex
prefers PtdIns(3)P. MTMR9 increased the enzymatic activity of
MTMR6 toward PtdIns(3,5)P2by over 30-fold, and enhanced the
activity toward PtdIns(3)P by only 2-fold. In contrast, MTMR9 in-
creased the activity of MTMR8 by 1.4-fold and 4-fold toward PtdIns
(3,5)P2and PtdIns(3)P, respectively. In cells, the MTMR6/R9 com-
plex significantly increases the cellular levels of PtdIns(5)P, the
product of PI(3,5)P2dephosphorylation, whereas the MTMR8/R9
complex reduces cellular PtdIns(3)P levels. Consequentially, the
MTMR6/R9 complex serves to inhibit stress-induced apoptosis
and the MTMR8/R9 complex inhibits autophagy.
tide profile is regulated by phospholipases, lipid kinases, and
phosphatases. Understanding the roles of inositol signaling has
expanded during the last decade and a number of these enzymes
have been shown to cause diseases when mutated (1). The tu-
mor-suppressor PTEN was discovered through positional cloning
as being mutated in several types of cancer (2, 3). PTEN was
subsequently shown to be a phosphatase, which dephosphor-
ylates phosphatidylinositol 3,4,5-trisphosphate to generate phos-
phatidylinositol 4,5-bisphosphate, an activity that is lost in
patients with PTEN mutations (4, 5). Mutations in the inositol
polyphosphate 5-phosphatase OCRL cause the X-linked disor-
der Lowe syndrome, which is associated with mental retardation,
blindness, and renal failure (6). Mutations in myotubularin cause
myotubular myopathy (7), and mutations in myotubularin-re-
lated protein 2 (MTMR2) and MTMR13 cause a form of
Charcot Marie Tooth disease type 4B, a demyelinating neuro-
degenerative disorder (8, 9).
The myotubularin family consists of 16 different proteins, 9
members of which possess catalytic activity (10, 11) and 7 mem-
bers that are inactive. Myotubularin proteins are not redundant
and have unique functions within cells by regulating a specific
pool of dephosphorylating phosphatidylinositol 3-phosphate
[PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns
(3,5)P2] (12–15). Varying tissue expression and subcellular lo-
calization play a role in determining the unique function of
nositol lipids play important roles in a variety of intracellular
signaling pathways. In response to stimuli, the phosphoinosi-
myotubularin proteins (16–21). One mechanism that regulates
the myotubularins is the formation of heterodimers between
catalytically active and inactive proteins. The interaction between
different myotubularin proteins has a significant effect on en-
zymatic activity. For example, the association of myotubularin
(MTM1) with MTMR12 results in a threefold increase in the 3-
phosphatase activity of MTM1, alters the subcellular localiza-
tion of MTM1 from the plasma membrane to the cytosol, and
attenuates the filopodia formation seen with MTM1 overex-
pression (21, 22). MTMR2 binds to MTMR5 via the coiled-coil
domains resulting in a three- to fourfold increase in 3-phospha-
tase activity and altered subcellular localization of MTMR2 (19).
MTMR2 also binds to MTMR13, resulting in a dramatic increase
in the catalytic activity of MTMR2 toward both PtdIns(3)P and
PtdIns(3,5)P2(23). In these examples, the binding of a catalyti-
cally inactive protein to a catalytically active protein resulted in
changes in activity and localization; hence, inactive myotubularin
proteins may serve a regulatory role. Mutations in both active and
inactive myotubularins are associated with diseases (8, 9), such as
myotubular myopathy, Charcot-Marie-Tooth disease, and others,
indicating that inactive myotubularin proteins are functionally
important. Based on these results, the working hypothesis is that
the enzymatically active myotubularin proteins dimerize with
enzymatically inactive myotubularin proteins, and the formation
of these heteromers can result in altered enzymatic activity and
subcellular localization. Myotubularin proteins can be grouped
into subfamilies based on homology. Closely related MTMR6,
MTMR7, and MTMR8 comprise such a subfamily, and MTMR9
is the sole member of another subfamily. Previous studies have
shown that MTMR9 binds to MTMR6 (24) and to MTMR7 (25).
PtdIns(3)P has been proposed to be essential in autophagy,
a conserved intracellular process for the degradation of cyto-
plasmic proteins or organelles. A number of human diseases,
including cancer and neurodegenerative disorders, are linked to
dysfunctions in autophagy (26). Autophagy has been demon-
strated to eliminate aggregated proteins in neurons (27). Previous
studies have shown that aggregated proteins have pathological
significance with respect to neurodegeneration, as removal of
these proteins in mouse models of spinocerebellar ataxia 1 and
Huntington disease correlates with reversal of symptoms (28, 29).
Type III PI3K, which generates PtdIns(3)P in mammalian cells,
forms a complex with Beclin 1 and controls autophagosome for-
mation (30). Little is known what role the synthesis and degra-
dation of PtdIns(3)P plays in autophagy. It was proposed that
Author contributions: J.Z., C.Z., J.M., and P.W.M. designed research; J.Z., C.Z., J.M., M.V.K.,
and M.P.W. performed research; J.Z., C.Z., J.M., M.V.K., P.W.M., and M.P.W. analyzed
data; and J.Z. and M.P.W. wrote the paper.
The authors declare no conflict of interest.
1J.Z. and C.Z. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: PHIL@dom.wustl.edu, czhang@
dom.wustl.edu, or firstname.lastname@example.org.
| June 12, 2012
| vol. 109
| no. 24
when autophagy is suppressed under nutrient-rich conditions, the
activityofPtdIns(3)P phosphatasesoverrides thatof typeIIIPI3K
(31). Most recently, two members of the MTMR family, Jumpy
(MTMR14) and MTMR3, have been shown to be involved in the
regulation of autophagy (32, 33). Knockdown of Jumpy enhances
autophagy under both nutrient and starvation conditions, whereas
a dominant-negative MTMR3 only increases autophagic activity
in the presence of nutrients, suggesting that the roles of the two
MTMRs in autophagy are different. Down-regulation of both
MTMR3 and MTMR14 facilitates initiation as well as the com-
pletion of autophagy, indicating that the local PtdIns(3)P level is
important for the entire autophagic process. Here, we demon-
the MTMR6/R9 complex that regulates PtdIns (3, 5)P2levels and
thereby affects apoptosis, the MTMR8/R9 complex down-regu-
lates the levels of PthIns(3)P and blocks the autophagic process.
Human MTMR9 Binds to MTMR8 and Increases the Stability of MTMR8.
We previously demonstrated that human MTMR6 and MTMR9
directly associate both in vitro and in cells (24). Formation of the
MTMR6/R9 complex increased MTMR6’s affinity for phospholi-
pids, catalytic activity, and protein stability. Functionally, the com-
plex inhibited apoptosis (24). To investigate whether the human
orthologs of MTMR8 and MTMR9 interact, the human MTMR8
cDNA (GenBank, NM_017677) and human MTMR9 cDNA
(GenBank,NM_015458) were cloned,as previously described(24).
HA-MTMR8 and FLAG-MTMR9 were coexpressed in HeLa cells
Both MTMR8 and MTMR9 were detected when either MTMR8
or MTMR9 was immunoprecipitated (Fig. 1A).
We next tested whether or not formation of the complex sta-
bilizes the proteins, possibly by decreasing the degradation rate.
The levels of MTMR8 were analyzed in cycloheximide-treated
HeLa cells in the presence or absence of MTMR9. Higher levels
of MTMR8 and slower degradation of MTMR8 are observed in
cells cotransfected with both proteins (Fig. 1B).
Catalytic Activity of MTMR6, MTMR6/R9, MTMR8, and MTMR8/R9. To
determine the effect of MTMR9 on the 3-phosphatase activity of
both MTMR6 and MTMR8, we determined the catalytic activity
for each using radio-labeled PtdIns(3*)P and PtdIns(3*,5)P2, by
measuring the release of [32P]-PO4. MTMR9 increased the en-
zymatic activity of MTMR6 toward PtdIns(3)P only about 2-fold,
whereas it enhanced the activity toward PtdIns(3,5)P2by over
30-fold. By contrast, MTMR9 increased MTMR8 activity 4-fold
and 1.4-fold toward PtdIns(3)P and PtdIns(3,5)P2, respectively
(Fig. 2 A and B). The cellular level of the product PtdIns(5)P was
elevated threefold when both MTMR6 and MTMR9 were coex-
pressed but no significant increase in the level of PtdIns(5)P was
seen by overexpression of MTMR8 plus MTMR9 (Fig. 2C),
consistent with the changes in enzymatic activity observed in the
in vitro assays. MTMR8exhibited relatively higher activity toward
PtdIns(3)P than MTMR6 or MTMR6 plus MTMR9 in vitro (Fig.
2A); however, no significant decrease was observed in the cellular
levels of PtdIns(3)P by overexpression of MTMR8, using an an-
tibody that specifically recognizes PtdIns(3)P. Transfection effi-
ciency was determined in a separate set of plates and found to be
greater than 95%. No significant change in PtdIns(3)P levels was
observed in cells overexpressing MTMR6 or MTMR6 plus
MTMR9 (Fig. 2D). These data are quantified in Fig. 2E, with 50
cells per cover-slip, and three cover-slips counted for each con-
dition. Spots larger than 1 nm were counted as one PI(3)P mol-
ecule. Only overexpression of MTMR8 plus MTMR9 altered the
cellular levels of PtdIns(3)P significantly, implying that the
MTMR6/R9 complex controls PtdIns(3,5)P2, but the MTMR8/
R9 complex determines PtdIns(3)P levels, thereby possibly af-
fecting different cellular functions. A moderate decrease was
observed in the level of PtdIns(3)P, with overexpression of
MTMR9 alone, suggesting that inactive MTMR9 altered these
levels through interactions with endogenous MTMR8.
Role of the MTMR8/R9 Complex in Autophagy. A number of studies
in Caenorhabditis elegans indicate that several myotubularins
have nonredundant roles in regulating PtdIns(3)P levels during
endocytosis (17, 34). It is likely that MTMR6 and MTMR8 have
distinct functions because of different substrate specificities and
specific subcellular localization. We have demonstrated that the
MTMR6/R9 complex protects cells from etoposide-induced ap-
optosis (24). However, the antiapoptosis effect was not seen with
overexpression of MTMR8 and MTMR9 (Fig. 2F). As our
in vitro studies suggested that the MTMR8/R9 complex controls
a cellular pool of PtdIns(3)P, we examined the cellular con-
sequences that result from both increasing and decreasing the
levels of MTMR8 and MTMR9 in cells.
PtdIns(3)P has been proposed to be essential in autophagy,
a conserved intracellular process for the degradation of cyto-
plasmic proteins or organelles. Overexpression of both MTMR8
and MTMR9 resulted in a significant increase in the level of p62,
a protein that is degraded in autophagosomes and is used to
monitor autophagy (35) (Fig. 3A). Knockdown of either
MTMR8 or MTMR9 alone had no effect on the level of p62 in
cells, whereas knockdown of both MTMR8 and MTMR9 sig-
nificantly reduced the level of p62 in HeLa cells, treated with
100 nM bafilomycin A1 for 3 h to inhibit fusion between auto-
phagosomes and lysosomes (Fig. 3B). The level of MTMR8
following RNAi of MTMR8 was 0.275, when the level of
MTMR8 was set at 1.0 in control siRNA, as determined using
RT-PCR. Very little effect was seen in the levels of p62 with
RNAi of MTMR6 alone or in combination with MTMR9
MTMR8 (HA)MTMR8+R9 (FLAG)
CHX (hr)0 1 2 3 5 7 0 1 2 3 5 7
1.01.20.21 0.08 0.010.021.00.8 0.24 0.090.100.08
FLAG-MTMR9 for 24 h. Cycloheximide (150 μg/mL) was added for the times indicated. The levels of MTMR8 and MTMR9 were detected by Western blotting with an
anti-HA or anti-FLAG antibody, respectively. β-Actin levels are shown as a loading control. MTMR8 band intensities relative to actin are indicated below each lane.
Interaction between MTMR8 and MTMR9. (A) HeLa cells were cotransfected with HA-MTMR8 and MTMR9-FLAG for 24 h, and proteins were immunopre-
| www.pnas.org/cgi/doi/10.1073/pnas.1207021109Zou et al.
(Fig. 3C). Thus, inactive MTMR9 regulates its individual binding
partners’ discrete functions. Up- or down-regulation of MTMR8
alone had no significant effect on autophagy, as measured by p62
levels, compared with controls (Fig. 3 A and B). Knocking down
MTMR9 alone led to a notable effect on autophagy compared
with control RNAi or vector (Fig. 3B), implying that there might
be other members of this MTMR subfamily involved in the
autophagy pathway that are also controlled by MTMR9: for
example, MTMR7. HeLa cells transfected with MTMR8 for
overexpression, followed by RNAi of MTMR8, show reduced
expression of MTMR8, compared with RNAi of vector alone
(Fig. 3D). The levels of MTMR8 protein were also further re-
duced using a combination of RNAi oligonucleotides targeting
both MTMR8 and MTMR9 (Fig. 3D, lanes 3 and 6). This finding
suggested that the formation of the MTMR8/R9 complex sta-
bilizes the proteins, as we have previously seen with the
MTMR6/R9 complex (24). Moreover, when HeLa cells
expressing HA-MTMR8 and endogenous MTMR9 were placed
in serum-free medium, the complex between these proteins was
completely dissociated by 2 h (Fig. 3E).
Another PtdIns(3)P binding autophagy factor, WIPI-1 (WD
repeat domain, phosphoinositide interacting 1), is recruited to
autophagic membranes in a PtdIns(3)P-dependent fashion (36).
The quantification of WIPI-1 protein accumulation can be used
to monitor mammalian autophagy (36). HeLa cells were trans-
fected with GFP-WIPI for 24 h, transfected with MTMR6,
MTMR8, MTMR6 plus MTMR9, or MTMR8 plus MTMR9
constructs for overexpression or RNAi constructs for knock-
downs. The percentage of cells displaying distinct WIPI-I puncta
was used to quantify the extent of autophagy. Knockdown of
MTMR8 plus MTMR9 significantly induced autophagy, which is
not seen in cells treated with RNAis of MTMR6, MTMR8,
k, min /mg protein
Normalized PI(5)P level
Number of PI(3)P spots per cell
Fraction of apoptotic cells
k, min /mg protein
levels. HeLa cells were transfected with vector (control), cotransfected with either MTMR6 plus MTMR9 or MTMR8 plus MTMR9, and PtdIns(5)P levels were
measured as previously described (45). Results are presented as relative mass compared with that in vector-transfected cells. (D) Overexpression of MTMR8
plus MTMR9 reduces levels of PtdIns(3)P in COS-7 cells. Cells were stained with anti-PI(3)P antibodies followed by anti-mouse Alexa568-conjugated antibodies.
Nuclei are visualized with DAPI. Cotransfection efficiency was determined in a separate set of plates, because of limited available fluorescence channels, and
was found to be greater that 95% in all cases. (Magnification: 63×.) (E) Quantification of immunofluorescence shown in D, expressed as an average number of
PI(3)P spots per cell normalized to the number seen in MTMR8/R9 expressing cells. (F) Overexpression of MTMR8/R9 has no effect on apoptosis. HeLa cells were
treated with the indicated constructs for 36 h, then with 100 µM etoposide to induce apoptosis. After 8 h, cells were treated with APOPercentage dye to
selectively stain apoptotic cells with dark spots. Percentage of apoptotic cells are counted as the number of darker cells out of every 100 cells (*P < 0.01, t test).
Enzymatic activity of MTMR6, MTMR6/R9, MTMR8, and MTMR8/R9 toward PtdIns(3)P (A) or PtdIns(3,5)P2(B). (C) Measurement of cellular PtdIns(5)P
Zou et al.PNAS
| June 12, 2012
| vol. 109
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MTMR9, or the MTMR6/R9 complex (Fig. 4 A and B). Strik-
ingly, overexpression of MTMR8 plus R9 abolished rapamycine-
induced autophagy (Fig. 4 C and D).
We describe here two members of a closely related subfamily of
active myotubularins, MTMR6 and MTMR8, both of which
partner with the same inactive myotubularin MTMR9. Previous
studies have shown an association between MTMR6 and
MTMR9 in mouse and C. elegans (24, 34). Complex formation
between MTMR6 and MTMR9 increases MTMR6’s affinity for
phospholipids, catalytic activity, protein stability, and complex
formation inhibits apoptosis (24). We demonstrated that the
association of MTMR9 with MTMR6 not only enhances the
enzymatic activity of MTMR6, but also determined its substrate
preference and thus the levels of its product. We have found that
the MTMR6/R9 complex controls PtdIns(3,5)P2levels and the
MTMR8/R9 complex determines PtdIns(3)P levels, thereby af-
fecting different cellular functions. We have shown that the
MTMR8/R9 complex functions to reduce autophagy and the
MTMR6/R9 complex inhibits apoptosis.
Endocytic membrane trafficking is critically dependent on the
local synthesis of PtdInd(3)P and PtdIns(3,5)P2(37). PtdIns(3)P is
III PI3K complex (38). Early endosome antigen 1, a protein es-
sential for endosome fusion, binds to Rab5-GTP and PtdIns(3)P
through the FYVE domain on the PIKfyve kinase. PtdIns(3)P is
subsequently converted into PtdIns(3,5)P2by PIKfyve on multi-
vesicular late endosomes, which is required for protein sorting as
well as controlling lysosome size (39). Although the mechanisms
involved in the synthesis of phosphoinositides at endosomes are
well understood, little is known about the lipid phosphatases that
degrade endosomal PtdIns(3)P and PtdIns(3,5)P2. It is likely that
myotubularin proteins, which dephosphorylate PtdIns(3)P and
PtdIns(3,5)P2 at the D3 position, are involved in membrane
trafficking. Overexpression of MTM1 leads to enlarged endo-
somal structures and delayed movement of the epidermal growth
factor receptor into the lysosome, similar tothe observation made
when PtdIns(3)P or PtdIns(3,5)P2is depleted by mutations in
PI3K or PIKfyve (40, 41). A recent study with RNAi depletion of
MTM1 also led to accumulation of the epidermal growth factor
receptor in distinct endosomes, despite the increased level of
phosphoinositide levels in endosomes may contribute to the dis-
ease phenotype when MTM1 is mutated. We plan to investigate
endocytosis process, because each complex controls a distinct
phosphatidylinositol pool and therefore may regulate different
MTMR9 seems to play a central role in the regulation of all
three active members of this subfamily. Although to date no dis-
ease has been associated with mutations in this subfamily, new
mutations are being uncovered all the time and the detailed study
of this subfamily will lead to a better understanding of the bio-
chemical eventsunderlyinghumandiseases causedbymutationsin
these proteins. MTMR9 expression has been correlated with
obesity in humans, as determined from a large number of gene-
based single-nucleotide polymorphisms (43). A replicated associ-
ation between obesity and a single-nucleotide polymorphism lo-
cated in the MTMR9 gene was demonstrated in a study comprised
of 1,011 obese and 2,171 control individuals, P = 10−7(43). In this
same report, transcription of MTMR9 in the rat hypothalamic
WB: MTMR8 (HA)
R6R8 R9R6+9 R8+9
Luc R6R9R6+9Luc R6R9 R6+9
BafA1 + inhibitors
MTMR8, MTMR9, and p62. A nonspecific band (*) is seen below MTMR9 in the vector and MTMR8 lanes. (B) Western blotting of p62 in HeLa cell extracts
transfected with the indicated RNAi constructs. (C) HeLa cells were transfected for 24 h with the indicated RNAis, then treated for 4 h with 100 nM Bafilomycin
A1 (BafA1), 10 µg/mL E64d, and 10 µg/mL pepstatin. Cell lysates were immunoblotted with anti-p62 and anti–β-actin, and p62 band intensities relative to actin
are indicated below each lane. (D) HeLa cells were transfected with HA-MTMR8 for 24 h, treated with the indicated RNAi constructs for another 24 h, then
analyzed by blotting with an antibody against HA tag. (E) The MTMR8 and MTMR9 complex dissociates during starvation induced autophagy. HeLa cells were
transfected with HA-MTMR8 for 36 h, followed by serum starvation for the indicated times. Extracts were immunoprecipitated with an antibody against
MTMR9andimmunoblottedwithan anti-HA antibody todetect MTMR8.The proteinlevels ofMTMR8andMTMR9duringstarvationareshown ininputpanels.
The MTMR8/R9 complex regulates autophagy. (A) HeLa cells were transfected with the indicated constructs. Western blotting was used to detect
| www.pnas.org/cgi/doi/10.1073/pnas.1207021109Zou et al.
region was induced by fasting and reduced by a high-fat diet. Be-
cause MTMR9 lacks phosphatase activity, it is likely that it inter-
acts with one of the active phosphatases to cause the obesity
phenotype. To define the regulatory role of MTMR9 in vivo, an
Materials and Methods
All experiments were performed at least three times.
Reagents and Chemicals. All chemicals and reagents, unless specifically noted,
were purchased from Sigma–Aldrich. [γ-32P]-ATP was purchased from
Cloning, Expression, and Purification of Human MTMR6 and MTMR9. Full-length
humanMTMR9was cloned as described previously(24). Constructs forhuman
MTMR6, MTMR8, and MTMR9 were expressed in Sf9 cells and protein was
purified, as described previously (24). Briefly, Sf9 cells were transfected with
the appropriate construct, and the recombinant flag-tagged proteins were
purified using FLAG M1 agarose affinity gel (Sigma-Aldrich) according to the
Cell Culture, Transfection, Immunoprecipitation, and Western Blotting. HeLa
cells were maintained in culture using 10% (vol/vol) FBS in DMEM. Unless
noted, transfection was conducted by using Lipofectamine 2000 (Invitrogen).
RNAi transfections were done using a Nucleofector kit (Amaxa). RNAi
duplexes (Ambion) used in transfections are as follows: control RNAi (lucif-
erase) duplex, sense 5′-CUUACGCUGAGUACUUCGAdTdT-3′; antisense, 5′-
UCGAAGUACUCAGCGUAAGDTdT-3′; MTMR6 RNAi (R6-1), sense, 5′-GGA-
AGTCAATGGCACTAATgg-3′; antisense, 5′-TTTAGTGCCATTGACTTCCaa-3′;
MTMR8 RNAi (Invitrogen; Stealth RNAi, catalog # HSS124669); MTMR9 RNAi,
5′-CAAAGGAGGTGGCTTTGA Tca-3′and5′-TCAAAGCCACCTCCTTTGgc-3′. The
specificity and efficacy were determined using quantitative reverse
transcription-PCR, with 70%, 70%, and 50% reduction upon the RNAi of
MTMR6, MTMR8, and MTMR9, respectively. No cross-reactivity was detected.
Immunoprecipitation and Western blotting were done as previously de-
Measurement of PtdIns(5)P Mass, 3-Phosphatase Activity, and Protein Stability.
The PtdIns(5)P mass assay was conducted as previously described (45) with
minor modifications. HeLa cells were transfected with control vector,
MTMR6/R9, and MTMR8/R9 constructs. Total cellular phosphatidylinositol
content was purified using a PIP mass purification kit (Echelon Bioscience).
For measurement of 3-phosphatase activity, 100 ng of purified, recombinant
MTMR protein was added to a reaction mixture containing trace amounts of
[32P]PtdIns(3)P and [32P]PtdIns(3,5)P2, prepared as described previously (24).
Substrate identity and purity were determined by HPLC analysis. Activity
is expressed as a rate constant using the equation [S] = [So]e−kt. Units are
min−1, and are graphed as min−1/mg protein. Because of the trace amount
of label incorporated into the substrate, a defined specific activity cannot be
determined. Protein stability was measured as previously described (24).
Immunofluorescence Microscopy. Twenty-four hours after transfection, HeLa
cells grown on cover-slips were fixed as described previously (45), washed with
Tris-buffered saline, and solubilized with 0.5% Triton X-100 in PBS. Antibodies
were diluted in PBS, containing 0.1% Triton X-100 and 5% BSA. Cells were in-
at 37 °C. Cover-slips were washed in PBS, and mounted in Prolong mounting
medium (Molecular Probes). Images weretaken with anOlympus IX70 inverted
microscope and processed with Metamorph software (Molecular Devices).
ACKNOWLEDGMENTS. We thank Dr. Nordheim (Tuebingen, Germany) for
the gifts of WIPI-1 expression vectors (36); Peter Nicholas and Cecil Buchanan
for their helpful assistance; and Dr. Shao-Chun Chang for invaluable advice.
This work was supported by National Institutes of Health Grant HL16634 (to
P.W.M.) and Children’s Discovery Institute Grant MD-II-2009-174 (to M.P.W.).
constructs for 24 h. Autophagy was assessed by measuring WIPI-1 puncta-formation by immunofluorescence. (Magnification: 63×.) (B) Results from a total of
500 cells were counted and the ratios of cells in puncta/nonpuncta status was determined. (*P < 0.01.) (C) Overexpression of the MTMR8/R9 complex sup-
presses autophagy. HeLa cells were transfected with GFP-WIPI-1 for 24 h, then transfected with the indicated constructs for an additional 24 h. Autophagy
was induced by rapamycine for 3 h and then was measured by immunofluorescence. (Magnification: 63×.) (D) The ratios of cells in puncta/nonpuncta status
was measured. Cotransfection efficiency was determined to be greater than 95% using a duplicate set of plates. (E) The hypothesized consequential effects of
complex formation between MTMR9 and either MTMR8 or MTMR6 are shown in a schematic diagram.
Knockdown of the MTMR8/R9 complex induces autophagy. (A) HeLa cells were transfected with GFP-WIPI-1 for 24 h, then treated with indicated RNAi
Zou et al. PNAS
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1. Majerus PW, York JD (2009) Phosphoinositide phosphatases and disease. J Lipid Res Download full-text
2. Li J, et al. (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in
human brain, breast, and prostate cancer. Science 275:1943–1947.
3. Steck PA, et al. (1997) Identification of a candidate tumour suppressor gene, MMAC1, at
4. Myers MP, et al. (1997) P-TEN, the tumor suppressor from human chromosome 10q23,
is a dual-specificity phosphatase. Proc Natl Acad Sci USA 94:9052–9057.
5. Maehama T, Dixon JE (1998) The tumor suppressor, PTEN/MMAC1, dephosphorylates
the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273:
6. Attree O, et al. (1992) The Lowe’s oculocerebrorenal syndrome gene encodes a protein
highly homologous to inositol polyphosphate-5-phosphatase. Nature 358:239–242.
7. Laporte J, et al. (1996) A gene mutated in X-linked myotubular myopathy defines
a new putative tyrosine phosphatase family conserved in yeast. Nat Genet 13:
8. Bolino A, et al. (2000) Charcot-Marie-Tooth type 4B is caused by mutations in the gene
encoding myotubularin-related protein-2. Nat Genet 25:17–19.
9. Senderek J, et al. (2003) Mutation of the SBF2 gene, encoding a novel member of the
myotubularin family, in Charcot-Marie-Tooth neuropathy type 4B2/11p15. Hum Mol
10. Alonso A, et al. (2004) Protein tyrosine phosphatases in the human genome. Cell 117:
11. Vergne I, Deretic V (2010) The role of PI3P phosphatases in the regulation of au-
tophagy. FEBS Lett 584:1313–1318.
12. Laporte J, et al. (1998) Characterization of the myotubularin dual specificity phos-
phatase gene family from yeast to human. Hum Mol Genet 7:1703–1712.
13. Clague MJ, Lorenzo O (2005) The myotubularin family of lipid phosphatases. Traffic 6:
14. Laporte J, Bedez F, Bolino A, Mandel JL (2003) Myotubularins, a large disease-asso-
ciated family of cooperating catalytically active and inactive phosphoinositides
phosphatases. Hum Mol Genet 12(Spec No 2):R285–R292.
15. Taylor GS, Dixon JE (2003) PTEN and myotubularins: Families of phosphoinositide
phosphatases. Methods Enzymol 366:43–56.
16. Kim SA, Taylor GS, Torgersen KM, Dixon JE (2002) Myotubularin and MTMR2, phos-
phatidylinositol 3-phosphatases mutated in myotubular myopathy and type 4B
Charcot-Marie-Tooth disease. J Biol Chem 277:4526–4531.
17. Xue Y, et al. (2003) Genetic analysis of the myotubularin family of phosphatases in
Caenorhabditis elegans. J Biol Chem 278:34380–34386.
18. Berger P, Schaffitzel C, Berger I, Ban N, Suter U (2003) Membrane association of
myotubularin-related protein 2 is mediated by a pleckstrin homology-GRAM domain
and a coiled-coil dimerization module. Proc Natl Acad Sci USA 100:12177–12182.
19. Kim SA, Vacratsis PO, Firestein R, Cleary ML, Dixon JE (2003) Regulation of my-
otubularin-related (MTMR)2 phosphatidylinositol phosphatase by MTMR5, a catalyti-
cally inactive phosphatase. Proc Natl Acad Sci USA 100:4492–4497.
20. Lorenzo O, Urbé S, Clague MJ (2005) Analysis of phosphoinositide binding domain
properties within the myotubularin-related protein MTMR3. J Cell Sci 118:2005–2012.
21. Nandurkar HH, et al. (2003) Identification of myotubularin as the lipid phosphatase
catalytic subunit associated with the 3-phosphatase adapter protein, 3-PAP. Proc Natl
Acad Sci USA 100:8660–8665.
22. Caldwell KK, Lips DL, Bansal VS, Majerus PW (1991) Isolation and characterization of
two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and ino-
sitol 1,3-bisphosphate. J Biol Chem 266:18378–18386.
23. Berger P, et al. (2006) Multi-level regulation of myotubularin-related protein-2
phosphatase activity by myotubularin-related protein-13/set-binding factor-2. Hum
Mol Genet 15:569–579.
24. Zou J, Chang SC, Marjanovic J, Majerus PW (2009) MTMR9 increases MTMR6 enzyme
activity, stability, and role in apoptosis. J Biol Chem 284:2064–2071.
25. Mochizuki Y, Majerus PW (2003) Characterization of myotubularin-related protein 7
and its binding partner, myotubularin-related protein 9. Proc Natl Acad Sci USA 100:
26. Levine B, Deretic V (2007) Unveiling the roles of autophagy in innate and adaptive
immunity. Nat Rev Immunol 7:767–777.
27. Yamamoto A, Cremona ML, Rothman JE (2006) Autophagy-mediated clearance of
huntingtin aggregates triggered by the insulin-signaling pathway. J Cell Biol 172:
28. Yamamoto A, Lucas JJ, Hen R (2000) Reversal of neuropathology and motor dys-
function in a conditional model of Huntington’s disease. Cell 101:57–66.
29. Zu T, et al. (2004) Recovery from polyglutamine-induced neurodegeneration in con-
ditional SCA1 transgenic mice. J Neurosci 24:8853–8861.
30. Shintani T, Klionsky DJ (2004) Autophagy in health and disease: A double-edged
sword. Science 306:990–995.
31. Noda T, Matsunaga K, Taguchi-Atarashi N, Yoshimori T (2010) Regulation of mem-
brane biogenesis in autophagy via PI3P dynamics. Semin Cell Dev Biol 21:671–676.
32. Vergne I, et al. (2009) Control of autophagy initiation by phosphoinositide 3-phos-
phatase Jumpy. EMBO J 28:2244–2258.
33. Taguchi-Atarashi N, et al. (2010) Modulation of local PtdIns3P levels by the PI phos-
phatase MTMR3 regulates constitutive autophagy. Traffic 11:468–478.
34. Dang H, Li Z, Skolnik EY, Fares H (2004) Disease-related myotubularins function in
endocytic traffic in Caenorhabditis elegans. Mol Biol Cell 15:189–196.
35. Bjørkøy G, et al. (2005) p62/SQSTM1 forms protein aggregates degraded by au-
tophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171:
36. Proikas-Cezanne T, Ruckerbauer S, Stierhof YD, Berg C, Nordheim A (2007) Human
WIPI-1 puncta-formation: A novel assay to assess mammalian autophagy. FEBS Lett
37. Lindmo K, Stenmark H (2006) Regulation of membrane traffic by phosphoinositide
3-kinases. J Cell Sci 119:605–614.
38. Nicot AS, Laporte J (2008) Endosomal phosphoinositides and human diseases. Traffic
39. Shisheva A (2008) PIKfyve: Partners, significance, debates and paradoxes. Cell Biol Int
40. Robinson FL, Dixon JE (2006) Myotubularin phosphatases: Policing 3-phosphoinosi-
tides. Trends Cell Biol 16:403–412.
41. Tsujita K, et al. (2004) Myotubularin regulates the function of the late endosome
through the gram domain-phosphatidylinositol 3,5-bisphosphate interaction. J Biol
42. Cao C, Backer JM, Laporte J, Bedrick EJ, Wandinger-Ness A (2008) Sequential actions
of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor
receptor trafficking. Mol Biol Cell 19:3334–3346.
43. Yanagiya T, et al. (2007) Association of single-nucleotide polymorphisms in MTMR9
gene with obesity. Hum Mol Genet 16:3017–3026.
44. Zou J, et al. (2012) The role of myotubularin-related phosphatases in the control of
autophagy and programmed cell death. Adv Enzyme Regul 52:282–289.
45. Zou J, Marjanovic J, Kisseleva MV, Wilson M, Majerus PW (2007) Type I phosphati-
dylinositol-4,5-bisphosphate 4-phosphatase regulates stress-induced apoptosis. Proc
Natl Acad Sci USA 104:16834–16839.
| www.pnas.org/cgi/doi/10.1073/pnas.1207021109 Zou et al.